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The gut microbiota (GM) exerts a strong influence over the host immune system and dysbiosis of this microbial community can affect the clinical phenotype in chronic inflammatory conditions. To explore the role of the GM in lupus nephritis, we colonized NZM2410 mice with Segmented Filamentous Bacteria (SFB). Gut colonization with SFB was associated with worsening glomerulonephritis, glomerular and tubular immune complex deposition and interstitial inflammation compared to NZM2410 mice free of SFB. With SFB colonization mice experienced an increase in small intestinal lamina propria Th17 cells and group 3 innate lymphoid cells (ILC3s). However, although serum IL-17A expression was elevated in these mice, Th17 cells and ILC3s were not detected in the inflammatory infiltrate in the kidney. In contrast, serum and kidney tissue expression of the macrophage chemoattractants MCP-1 and CXCL1 were significantly elevated in SFB colonized mice. Furthermore, kidney infiltrating F4/80+CD206+ M2-like macrophages were significantly increased in these mice. Evidence of increased gut permeability or “leakiness” was detected in SFB colonized mice. Finally, the intestinal microbiome of SFB colonized mice at 15 and 30 weeks of age exhibited dysbiosis when compared to uncolonized mice at the same time points. Both microbial relative abundance as well as biodiversity of colonized mice was found to be altered. Collectively, SFB gut colonization in the NZM2410 mouse exacerbates kidney disease, promotes kidney M2-like macrophage infiltration and overall intestinal microbiota dysbiosis.

Recent studies implicate innate immunity to systemic lupus erythematosus (SLE) pathogenesis. Toll-like receptor (TLR)8 is estrogen-regulated and binds viral ssRNA to stimulate innate immune responses, but recent work indicates that microRNA (miR)-21 within extracellular vesicles (EVs) can also trigger this receptor. Our objective was to examine TLR8 expression/activation to better understand sex-biased responses involving TLR8 in SLE. Our data identify an estrogen response element that promotes STAT1 expression and demonstrate STAT1-dependent transcriptional activation of TLR8 with estrogen stimulation. In lieu of viral ssRNA activation, we explored EV-encapsulated miR-21 as an endogenous ligand and observed induction of both TLR8 and cytokine expression in vitro. Moreover, extracellular miR detection was found predominantly within EVs. Thus, just as a cytokine or chemokine, EV-encapsulated miR-21 can act as an inflammatory signaling molecule, or miRokine, by virtue of being an endogenous ligand of TLR8. Collectively, our data elucidates a novel innate inflammatory pathway in SLE.

Background High mobility group box one protein (HMGB1) is a chromatin associated protein, which in response to stress or injury translocates from the nucleus to the extracellular milieu to serve as an alarmin. HMGB1 has a remarkable ability to form complexes with proinflammatory molecules. This is underscored by the emerging role of HMGB1 in rheumatic diseases such as systemic lupus erythematosus (SLE), where the presence of HMGB1 in complex with endogenous nuclear components such as dsDNA or nucleosomes was shown to play an important role in breaking immune tolerance against nuclear antigens.1 Although the function of HMGB1 is in-part determined by the complexes it forms with other molecules, structural modifications in the HMGB1 polypeptide that may regulate complex formation have not been described. Objectives In this study we investigated the presence of HMGB1 in large protein complexes (HMGB1c) in human plasma. The objectives of this study were to isolate and characterise HMGB1c as well as to determine the mechanism of its formation. Methods We examined the presence of HMGB1c in plasma from SLE patients and healthy controls using semi-denaturating detergent agarose gel electrophoresis (SDD-AGE) folowed by Western blot analysis. Immunoblotting, coimmunoprecipitation, confocal microscopy, and mass spectrometry were used to detect and characterise novel high molecular weight HMGB1 variants in vitro as well as in cell lines and primary cells. Mechanisms of HMGB1c formation were delineated via mass spectrometry, RNA interference, and in vitro enzyme reactions. Results In this study we note the presence of high molecular weight, denaturing resistant HMGB1 protein complexes (HMGB1c) that were present in the plasma of SLE patients and to a much lesser extent, healthy subjects. Similarly, HMGB1c were induced when cells were incubated with endotoxin or alum. Here we report that HMGB1c formation is catalysed by the calcium-activated protein crosslinking enzyme transglutaminase-2 (TG2). HMGB1-TG2 interaction was demonstrated via coimmunoprecipitation as well as by confocal microscopy after co-transfection of cells with plasmids encoding fluorescent-tagged HMGB1 and TG2 constructs. Moreover, HMGB1c formation was suppressed in cells by TG2 siRNA. Crosslink site mapping and analysis by mass spectrometry revealed that HMGB1 can be crosslinked to TG2 as well as a number of additional proteins, including human autoantigens. Conclusions TG2 catalyses the formation of high molecular weight HMGB1 protein complexes. Given the immunoadjuvant properties of HMGB1 and the implication of TG2-mediated protein complex formation as a possible mechanism by which immune tolerance can be broken to self-molecules,2 these findings have significant physiological implications for the role of HMGB1 in cellular stress responses and innate immunity in lupus. References [1] Urbonaviciute V, Furnrohr BG, Meister S, Munoz L, Heyder P, De MF, et al. Induction of inflammatory and immune responses by HMGB1-nucleosome complexes: implications for the pathogenesis of SLE. J Exp Med2008;205:3007–3018. [2] Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med1997;3:797–801. Disclosure of Interest None declared

Genome-wide association studies (GWASs) have been instrumental in understanding complex phenotypic traits. However, they have rarely been used to understand lineage-specific pathways and functions that contribute to the trait. In this study, by integrating lineage-specific enhancers from mesenchymal and myeloid compartments with bone mineral density loci, we were able to segregate osteoblast- and osteoclast (OC)-specific functions. Specifically, in OCs, a PU.1-dependent transcription factor (TF) network was revealed. Deletion of PU.1 in OCs in mice resulted in severe osteopetrosis. Functional genomic analysis indicated PU.1 and MITF orchestrated a TF network essential for OC differentiation. Several of these TFs were regulated by cooperative binding of PU.1 with BRD4 to form superenhancers. Further, PU.1 is essential for conformational changes in the superenhancer region of Nfatc1. In summary, our study demonstrates that combining GWASs with genome-wide binding studies and model organisms could decipher lineage-specific pathways contributing to complex disease states., Genetics: Cellular fate dictates osteoporosis risk Genetic variation in non-coding regions of DNA could raise osteoporosis risk by affecting osteoclast differentiation. Osteoporosis occurs when the normal process of bone remodeling by osteoblasts and osteoclasts falls out of balance. Genome-wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) associated with osteoporosis, but how these affect specific cell types was unclear. Sudarshana Sharma and Michael Ostrowski at the Medical University of South Carolina and colleagues wondered if variations in non-coding ‘enhancer’ regions of DNA, might shed light on the molecular underpinnings of osteoporosis. So, they overlaid SNPs associated with reduced bone mineral density onto enhancers in mesenchymal and myeloid cells—the precursors of osteoblasts and osteoclasts—identifying a transcription factor network in myeloid cells that drives the differentiation of osteoclasts. When this was disrupted in mice, severe defects in osteoclast differentiation and function resulted.

Since being discovered over half a century ago, mesenchymal stem cells (MSCs) have been investigated extensively to characterize their cellular and physiological influences. MSCs have been shown to possess immunosuppressive capacity through inhibiting lymphocyte activation/proliferation and proinflammatory cytokine secretion while simultaneously demonstrating limited allogenic reactivity, which subsequently led to the evaluation of therapeutic feasibility to treat inflammatory diseases. Although regulatory constraints have restricted MSC development pharmacologically, limited clinical studies have shown encouraging results using MSC infusions to treat systemic lupus erythematosus (SLE); but, more trials will have to be performed to conclusively determine the clinical efficacy of MSCs to treat SLE. Moreover, there are some data to suggest that MSCs possess tumorigenic potential and that the immunosuppressive influence can be dramatically affected by both donor variability and ex vivo expansion. Given that recent studies have found that the immunosuppressive effects of MSCs are a result, at least in part, to extracellular vesicle (EV) secretion, the use of MSC-derived EVs has been suggested as a cell-free therapeutic alternative. Despite the positive data observed using EVs isolated from human MSCs to suppress inflammatory responses in vitro and in inhibiting autoimmune disease pathogenesis in preclinical work, there are no studies to date examining EVs from MSCs to treat SLE in humans or animal models. Considering that EVs are not subject to the strict regulatory constraints of stem cell-based pharmacological development and are more readily standardized with regard to industrial-scale production and storage, this review outlines the anti-inflammatory biology of MSCs and the scientific evidence supporting the potential use of EVs derived from human MSCs to treat patients with SLE.

Background and aims We have previously demonstrated that toll-like receptor (TLR)7 and TLR8 are significantly up-regulated in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus (SLE) patients and can be further induced with oestrogen treatment. It has recently been shown that specific micro-RNA (miR) sequences packaged in extracellular vesicles can stimulate these receptors in addition to the conventional activation by binding single-stranded RNA of viral origin. The aim of this study was to explore the feasibility of using miR antagonists to block TLR7 and TLR8-mediated inflammatory pathways. Methods Human-mouse chimaeras were generated by adoptively transferring PBMCs from active SLE patients into immunodeficient NOD-scid IL-2rγ (null) mice using a modified protocol that we previously established in Sjojren’s syndrome. Prior to transfer, SLE patient PBMCs were treated either with a cocktail of locked nucleic acid antagonists targeting several miRs or nonsense, scrambled controls. At 21 days post-transfer, blood was collected for flow cytometry and cytokine analysis; tissues were processed for histopathological examination by H and E and immunohistochemistry. Results The phenotypic characteristics of various immune cells were similar in both experimental groups; however, inhibition with miR antagonists reduced levels of human IL-2, IL-6, IL-10, and TNF-α relative to scramble (control) treatment. Histopathological analysis revealed that miR antogonists inhibited the robust responses detected with control treatment in the small intestine, liver, and kidney. Further characterisation of infiltrates confirmed the presence human CD3+ T-cells. Conclusions These data establish a novel model to study SLE and provide experimental evidence that TLR7 and TLR8 targeted miR antagonists have therapeutic potential in SLE.

Background Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease affecting multiple organs and displaying a heavy female predominance during reproductive years. Our previous studies have demonstrated that toll-like receptor (TLR)7 and TLR8 are significantly up-regulated in peripheral blood mononuclear cells (PBMCs) of SLE patients and induced with estrogen treatment. While the conventional role of TLR7 and TLR8 involves binding to single-stranded RNA of viral origin to stimulate innate inflammatory responses, recent work has identified specific micro-RNAs (miRs) capable of activating these receptors that are packaged and secreted in exosomes. Objectives The goal of this study was to explore the therapeutic potential of using a cocktail of miR antagonists to block TLR7 and TLR8 inflammatory pathways induced by extracellular vesicle (EV)-derived miRs in SLE. Methods PBMCs from active SLE patients were adoptively transferred into immunodeficient NOD-scid IL-2rγ (null) mice to produce chimeras using a similar protocol we have previously established for Sjojren9s syndrome. PBMCs were treated with a cocktail of locked nucleic acid miR antagonists or nonsense, scrambled RNA control prior to injection. Blood was collected for both flow cytometry and cytokine analysis and tissues were processed for histopathological examination by H&E and immunohistochemistry. Results 21 days after adoptive transfer, human T-cells (CD4+ and CD8+), B-cells, monocytes, and NK cells were all successfully recovered from whole blood of chimeric mice at similar levels in both treatment groups. However, miR inhibition reduced levels of human IL-2, IL-6, IL-10, and TNF-α when compared to scramble (control) treatment. Although histopathological analysis revealed little to no inflammation in the skin and ear, miR inhibition blocked the robust responses detected in the small intestine, liver, and kidney with scramble RNA (control) treatment. Immunohistochemical analysis of the infiltrates confirmed the presence human CD3+ T-cells in these tissues and further demonstrated the suppression of inflammation following miR inhibition. Conclusions These results establish a novel chimeric model to study SLE and provide a platform to further explore the therapeutic efficacy of suppressing EV-encapsulated miRs that bind to TLR7 and TLR8 to induce autoimmune-mediated inflammatory responses. References Fabbri M, Paone A, Calore F, Galli R, Gaudio E, Santhanam R, et al. MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response. Proceedings of the National Academy of Sciences of the United States of America 2012, 109(31): E2110–2116. Young NA, Wu LC, Burd CJ, Friedman AK, Kaffenberger BH, Rajaram MV, et al. Estrogen modulation of endosome-associated toll-like receptor 8: an IFNalpha-independent mechanism of sex-bias in systemic lupus erythematosus. Clinical immunology 2014, 151(1): 66–77. Young NA, et al. A chimeric human-mouse model of Sjogren9s syndrome. Clinical immunology, 2014;156:1–8. Acknowledgement We would like to extend our appreciation to all the volunteers that participated in this study and thank the Center for Clinical and Translational Science/Research Match program at The Ohio State University Wexner Medical Cetner and the American Red Cross. Disclosure of Interest None declared

Background Recent studies suggest that innate immune responses may play a significant pathological role in systemic lupus erythematosus (SLE). TLR8 is an innate immune system receptor that stimulates inflammation by recognizing and binding to single-stranded viral RNA sequences, but recent work has shown that miR-21 can also act as an endogenous TLR8 ligand in carcinogenesis via exosomes. Previously, we demonstrated that estrogen can lower the threshold of immune cell activation and lead to enhanced TLR8 expression, especially in the presence of synthetic TLR8 agonist stimulation. Objectives Our goal was to better characterize the estrogen-mediated signaling pathway in SLE leading to TLR8 up-regulation and to explore endogenous ligand stimulation. We hypothesized that estrogen stimulation of STAT1 could lead to transcriptional activation of TLR8, which could be stimulated by miR-21. Methods Cells were treated with estrogen and collected for RT-PCR or Western blot analysis. ChIP-seq identified putative transcriptional binding sites following estrogen stimulation and EMSA confirmed DNA-protein complex formation with these sequences. STAT1 and estrogen receptor-α (ERα) expression were blocked by siRNA. Liposomal complexes containing fluorescently-labeled miR-21-Cy3 (red) were incubated in vitro ; cells were imaged in real time using fluorescent microscopy and TLR8 expression was measured by RT-PCR. Results Estrogen stimulated STAT1 expression at the transcript level and enhanced phosphorylation of STAT1 was observed at the protein level in primary human cells and cell lines. CHiP-seq and EMSA analysis in cells stimulated with estrogen identified a putative response element for ERα to promote STAT1 expression and also confirmed STAT1-mediated transcriptional activation of TLR8. Using siRNA to target either ERα or STAT1, estrogen-medicated TLR8 induction was inhibited. To examine the role of endogenous ligand, miR-21 was detected in exosomes isolated from SLE serum and TLR8 expression was induced in vitro by stimulating cells with synthetically produced pseudoexosomes containing miR-21-Cy3. Conclusions Therefore, similar to a cytokine or chemokine, exosome-encapsulated miR-21 can function as an inflammatory signaling molecule, or miRokine, by virtue of being an endogenous ligand for TLR8. Collectively, our data elucidates a novel innate inflammatory pathway in SLE by showing that estrogen-mediated expression of STAT1 promotes TLR8 expression, which can then be triggered by miR-21. Since this may significantly influence autoimmune inflammation, a future focus of this work will be to identify additional TLR agonists and to block exosomal miRokine signaling using antagomiR silencing. References Fabbri, M., et al., MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response. Proc Natl Acad Sci USA, 2012. 109(31): p. E2110-6. Young, N.A., et al., Estrogen modulation of endosome-associated toll-like receptor 8: an IFNalpha-independent mechanism of sex-bias in systemic lupus erythematosus. Clin Immunol, 2014. 151(1): p. 66-77. Acknowledgements Funding was provided through the Wexner Medical Center at The Ohio State University. Disclosure of Interest None declared

Background Systemic Lupus Erythematosus (SLE) is a devastating multi-organ autoimmune disease displaying an overwhelming female predilection. Estrogen (E2) influences gene expression by binding to estrogen receptors (ERα and ERβ) and has been suggested in SLE pathogenesis. Further, E2 has been shown to regulate Signal Transducer and Activator of Transcription (STAT) 1 and STAT4 function, and to have known associations with SLE. Toll-like receptor 8 (TLR8) has also been implicated in SLE and is a potent inflammatory mediator. Objectives In this study, our aim was to demonstrate the E2-mediated induction of STAT1 and STAT4 for the first time in primary human peripheral blood mononuclear cells (PBMCs) and mechanistically characterize the pathway that is operative in this induction. Methods Putative estrogen response elements (EREs) in the human genome were identified from ChIP-seq data of MCF-7 cells stimulated with E2 for 45 min. PBMCs isolated from SLE patients and healthy controls were treated with a physiological dose of E2. Radio-labeled probes corresponding to STAT1 DNA binding regions were used in EMSA analysis to examine DNA-protein complex formation in a human monocytic cell line (THP-1), which lacks detectable IFNα expression. In the presence of E2, siRNA was used to block ERα, IFNα, or STAT1 expression in primary human macrophages or THP-1 cells. Results STAT1 is located just upstream of STAT4 and ChIP-seq data revealed an intragenic ERE binding peak within the STAT1 locus with E2 treatment, suggesting that ERα may be promoting the expression of both genes (Fig 1). To demonstrate E2-mediated induction of STAT1 and STAT4, primary human PBMCs from SLE patients and healthy controls were treated with a physiological dose of E2. Expression of STAT1 and STAT4 was significantly induced relative to untreated controls. Given that we have previously observed that TLR8 expression is higher in SLE and induced with E2 treatment, a bona fide STAT1 binding region proximal to the TRL8 gene was used in EMSA analysis and showed enhanced DNA-protein complex formation with E2 stimulation. Moreover, siRNA blocking STAT1 significantly reduced E2-mediated TLR8 induction. In primary human cells and in a hematopoietic cell line, siRNA blocking ERα inhibited STAT1 and STAT4 induction with E2 stimulation, while targeting IFNα had no effect. Conclusions E2 can stimulate the expression of STAT1 and STAT4 in PBMCs and subsequently could lead to the pathogenesis observed in SLE due to their potent signal transducing effects over the immune system. Additionally, the identification of molecular targets to inhibit the TLR8 inflammatory pathway presents a significant therapeutic opportunity. Collectively, our results indicate that STAT1 and STAT4 may contribute to the E2-mediated sex-bias observed in autoimmunity through ERα signaling and may potentially be ideal targets in future therapy to treat SLE. Acknowledgements We would like to extend our appreciation to all the volunteers that participated in this study. Our research would not be possible without the time and effort they put forth. We would also like to thank the Center for Clinical and Translational Science9s Research Match program at OSUWMC and the American Red Cross. Both organizations were instrumental in the progress of our research. Funding for this work provided through NIDDK K23DK059850, NIEHS R00ES019918, and the Wexner Medical Center at The Ohio State University. Funding for the Center for Clinical and Translational Science provided through CTSA grant number UL1TR000090. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3909